The Neisseria gonorrhoeae accessory genome and its association with the core genome and antimicrobial resistance

The bacterial accessory genome provides the genetic flexibility needed to facilitate environment and host adaptation. In Neisseria gonorrhoeae, known accessory elements include plasmids which can transfer and mediate antimicrobial resistance (AMR); however, chromosomal accessory genes could also play a role in AMR. Here, the gonococcal accessory genome was characterized using gene-by-gene approaches and its association with the core genome and AMR were assessed. The gonococcal accessory gene pool consisted of 247 genes, which were mainly genes located on large mobile genetic elements, phage associated genes, or genes encoding putative secretion systems. Accessory elements showed similar synteny across genomes, indicating either a predisposition for particular genomic locations or ancestral inheritance that are conserved during strain expansion. Significant associations were found between the prevalence of accessory elements and core genome multi-locus sequence types (cgMLST), consistent with a structured gonococcal population despite frequent horizontal gene transfer (HGT). Increased prevalence of putative DNA exchange regulators was significantly associated with AMR, which included a putative secretion system, methyltransferases and a toxin-antitoxin system. Although frequent HGT results in high genetic diversity in the gonococcus, we found that this is mediated by a small gene pool. In fact, a highly organized genome composition was identified with a strong association between the accessory and core genome. Increased prevalence of DNA exchange regulators in antimicrobial resistant isolates suggests that genetic material exchange plays a role in the development or maintenance of AMR. These findings enhance our understanding of gonococcal genome architecture and have important implications for gonococcal population biology. IMPORTANCE The emergence of antimicrobial resistance (AMR) against third generation cephalosporins in Neisseria gonorrhoeae is a major public health concern, as these are antibiotics of last resort for the effective treatment of gonorrhea. Although the resistance mechanisms against this class of antibiotics have not been entirely resolved, resistance against other classes of antibiotics, such as tetracyclines, is known to be mediated through plasmids, which are known gonococcal extra-chromosomal accessory elements. A complete assessment of the chromosomal accessory genome content and its role in AMR has not yet been undertaken. Here, we comprehensively characterize the gonococcal accessory genome to better understand genome architecture as well as the evolution and mechanisms of AMR in this species

Emergence of a Neisseria gonorrhoeae clone with reduced cephalosporin susceptibility between 2014 and 2019 in Amsterdam, The Netherlands, revealed by genomic population analysis

BACKGROUND: Emerging resistance to cephalosporins in Neisseria gonorrhoeae (Ng) is a major public health threat, since these are considered antibiotics of last resort. Continuous surveillance is needed to monitor the circulation of resistant strains and those with reduced susceptibility. OBJECTIVES: For the purpose of epidemiological surveillance, genomic population analysis was performed on Ng isolates from Amsterdam with a focus on isolates with reduced susceptibility to ceftriaxone. METHODS: WGS data were obtained from 318 isolates from Amsterdam, the Netherlands between 2014 and 2019. Isolates were typed according to MLST, Ng Multi-Antigen Sequence Typing (NG-MAST) and Ng Sequence Typing for Antimicrobial Resistance (NG-STAR) schemes and additional resistance markers were identified. Phylogenetic trees were created to identify genetic clusters and to compare Dutch and non-Dutch MLST7827 isolates. RESULTS: MLST7363 and MLST1901 were the predominant strains having reduced susceptibility to ceftriaxone during 2014-16; MLST7827 emerged and dominated during 2017-19. NG-STAR38 and NG-MAST2318/10386 were predominant among MLST7827 isolates. MLST7827 reduced susceptibility isolates carried a non-mosaic 13.001 penA allele with an A501V mutation and porB1b G120K/A121D mutations, which were lacking in susceptible MLST7827 isolates. Phylogenetic analysis of all publicly available MLST7827 isolates showed strong genetic clustering of Dutch and other European MLST7827 isolates. CONCLUSIONS: MLST7827 isolates with reduced ceftriaxone susceptibility have emerged during recent years in Amsterdam. Co-occurrence of penA A501V and porB1b G120K/A121D mutations was strongly associated with reduced susceptibility to ceftriaxone. Genetic clustering of Dutch and other European MLST7827 isolates indicates extensive circulation of this strain in Europe. Close monitoring of the spread of this strain having an alarming susceptibility profile is needed

Within-Host Genetic Variation in Neisseria gonorrhoeae over the Course of Infection

Knowledge of within-host genetic variation informs studies on transmission dynamics. We studied within-host genetic variation in Neisseria gonorrhoeae over the course of infection and across different anatomical locations. Isolates were obtained during a clinical trial, and isolates from consecutive time points reflected persistent infections after treatment failure. We compared sequence types (STs) and recombination unfiltered- and filtered core genome single nucleotide polymorphism (SNP) distances in 65 within-host isolate pairs from the same anatomical location over time-obtained with a median interval of 7 days-and 65 isolate pairs across different anatomical locations at one time point. Isolates with different Multi-Locus Sequence Types (MLST), NG-Sequence Types for Antimicrobial Resistance (NG-STAR) and NG-Multi Antigen Sequence Types (NG-MAST) had a median of 1466 recombination filtered SNPs, whereas a median of 1 SNP was found between isolates with identical STs or a different NG-MAST only. The threshold for differentiating between strains was set at 10 recombination filtered SNPs, showing that isolates from persistent infections could have different NG-MASTs. Antibiotic pressure applied through treatment did not lead to an increase in genetic variation in specific genes or in overall extent of variation, compared to variation across anatomical locations. Instead, within-host genetic variation was proposedly driven by the host immune response, as it was concentrated in genomic regions encoding surface exposed proteins involved in host-microbe interaction. Ultimately, 15/228 (6.5%) between-host pairs contained a single strain, suggesting between-host transmission. However, patient reported data are needed to differentiate within-host persistence from between-host transmission. IMPORTANCE Understanding transmission dynamics of Neisseria gonorrhoeae (Ng) is based on the identification of transmission events. These can be identified by assessing genetic relatedness between Ng isolates, expressed as core genome SNP distances. However, a SNP threshold to differentiate between strains needs to be defined, using knowledge on within- and between-host genetic variation. Here, we assessed within-host genetic variation, using a unique set of within-host Ng isolates from the same anatomical location over time or across different anatomical locations at one time point. The insights in genetic variation that occurred during the infection period contribute to the understanding of infection dynamics. In addition, the obtained knowledge can be used for future research on transmission dynamics and development of public health interventions based on bacterial genomic data

Increased clonality among Neisseria gonorrhoeae isolates during the COVID-19 pandemic in Amsterdam, the Netherlands

Distancing measures during the COVID-19 lockdown led to a temporary decrease of casual sex partners among clients of the Centre for Sexual Health (CSH) in Amsterdam, the Netherlands. We investigated the effect of this change on the genotypic and phenotypic distribution of Neisseria gonorrhoeae (Ng) isolates from CSH patients. From each Ng-positive patient we sequenced one isolate, resulting in 322 isolates which constituted two groups: 181 isolates cultured from 15 January to 29 February 2020 (before the first lockdown) and 141 cultured from 15 May to 30 June 2020 (during the first lockdown). Patient characteristics showed significantly more symptomatic patients and significantly fewer reported sex partners during the lockdown. Phenotypic data showed an increase in low-level azithromycin resistance and ceftriaxone susceptibility during the lockdown, and this remained after the study period. The diversity in sequence types (STs) decreased slightly during the lockdown. A shift occurred from ST 8156 being predominant before lockdown to ST 9362 during lockdown and a remarkably low median SNP distance of 17 SNPs was found between ST 9362 isolates obtained during lockdown. These findings reflect restricted travel and the change in sexual behaviour of CSH clients during the lockdown, with a potentially increased local transmission of the ST 9362 strain during this period, which led to genotypic and phenotypic changes in the Ng population. This shows that public health measures have far-reaching consequences and should be considered in the surveillance of other infectious diseases